The present invention relates to an apparatus for casting electrophoresis gels and performing electrophoresis for separation and analysis of DNA molecules, proteins and other charged molecules. The present invention also relates to a method of casting electrophoresis gels.
Prior art electrophoresis assemblies include vertical and horizontal arrangements constructed of impermeable, non-conducting plates of glass or plastic between which electrophoresis gels are molded and contained during electrophoresis. Horizontal assemblies are less commonly used due to a number of inherent disadvantages and difficulties in casting, loading and electrophorescing horizontal gels. For instance, sample wells of horizontal assemblies that serve as sample loading sites are typically constructed of the same materials used to form gels. Sample wells are defined by molding well walls into a thickness of a gel. This molding technique is convenient and enables samples wells to be formed simultaneously with or immediately after the casting of gels. However, well walls formed of gel matrix materials contain the same electrolytic buffer ions as gels and are capable of conducting electrical current, which often results in problems with the introduction of samples into gels. When electrical current is applied to assemblies for sample injection, electrolytic buffer ions in well walls compete with similar ions in samples, causing the introduction of sample ions into gels to occur slowly. This effect produces broader sample peaks and limits the ability of the electrophoresis assemblies to resolve molecules of different sizes. In addition, well walls formed of gel materials are not mechanically reliable and susceptible to breakage and tearing during gel casting and sample loading.
Vertical electrophoresis assemblies well known in the art avoid problems associated with samples wells formed of gel materials by using non-conducting well dividers, often referred to as xe2x80x9cshark""s tooth combsxe2x80x9d, constructed of durable materials. A non-conducting comb, such as that disclosed in U.S. Pat. Nos. 4,883,577, 4,909,918 and 5,164,065, is inserted between the assembly plates and placed in a plane of a gel to provide a non-conducting, xe2x80x9chard wellxe2x80x9d sample loading site. Such non-conducting combs are difficult and inconvenient to insert between the plates of horizontal electrophoresis assemblies. When used in horizontal assemblies, such combs do not provide sample wells that are cable of containing samples by gravity. Therefore, sample wells of horizontal assemblies are invariably formed from gel materials and are electrically conductive.
Another disadvantage of horizontal assemblies is that they require external biasing mechanisms to maintain the assembly plates and buffer reservoirs in precise relationship during the casting and electrophorescing of gels. These biasing mechanisms are typically built with very high tolerances and require continual and difficult manual adjustment. Often external biasing systems work in conjunction with a base or other substrate, wherein the assembly plates and reservoirs are maintained in precise relationship by connection of the assembly components to the base. As disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971, a horizontal gel assembly is connected to a base by means of adjustable clamps. Uniform and effective biasing requires accurate and frequent manual adjustment of the clamps. In addition, since external pressure is required to cast gels in such a horizontal assembly, the assembly cannot be removed from its casting position in the base without the risk of the assembly coming apart.
The base disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971 also serves as a water jacket for temperature control of gel during electrophoresis. Such a water jacket cannot be constructed of a sufficiently transparent material to permit the use of photoinitiators, such as riboflavin or benzoin methyl ether, to initiate gel polymerization, and is limited to the use of chemical initiators. Chemically-initiated gel polymerization takes several hours, while photo-initiated gels polymerize in only a few minutes. In addition, the bases and water jackets as disclosed in U.S. Pat. Nos. 5,137,613 and 5,228,971 are expensive and difficult to manufacture.
Prior art horizontal assemblies are also prone to leak fluid from buffer reservoirs. In addition, horizontal assemblies are often not of sufficient size to accommodate various high performance electrophoretic techniques, such as DNA sequencing and DNA fragment size analysis. The technique of DNA sequencing and fragment analysis require longer electrophoresis assemblies on the order of 50 cm or longer. Longer horizontal assemblies require a greater number of amp-hours of electrophoretic current and, hence, a greater supply of buffer ions for a single electrophoretic run. Therefore, buffer reservoirs must be larger in horizontal assemblies used in these techniques to provide a sufficient supply of buffer ions to maintain a consistent electric current for the duration of an electrophoretic run. Buffer reservoirs of prior art horizontal assemblies as those disclosed in U.S. Pat. Nos. 5,137,613, 5,228,971 and 5,242,568, cannot be significantly enlarged without such designs becoming difficult to handle. In addition, such prior art buffer reservoirs are sealed only by means of gaskets, and an increase in reservoir size would render such buffer reservoirs more prone to leak fluid.
Therefore, it is desirable to provide a horizontal electrophoresis assembly for use in high performance electrophoretic techniques, such as DNA sequencing and DNA fragment analysis, that overcomes the limitations and disadvantages of prior art assemblies. It is desirable that the horizontal electrophoresis assembly include sample wells with well walls constructed of a durable and electrically non-conductive material to help ensure uniform and consistent sample injection into a gel. It is also desirable that the horizontal assembly is structured and constructed such that gels are optically accessible to permit photopolymerization and optical detection of separated molecules. The horizontal assembly that provides flexibility to increase the size of buffer reservoirs is also desirable. In addition, it is desirable that the structure and construction of the horizontal assembly facilitate formation of leak-proof seals between assembly components.
The invention provides an electrophoresis cassette to cast electrophoresis gels and to separate and analyze molecular components by electrophoresis. The invention also provides a method of casting electrophoresis gels.
A first embodiment of the invention includes an electrophoresis cassette comprising a top plate assembly seated on a bottom plate with a continuous spacer therebetween to define a thickness of the electrophoresis cassette and a molding space. The spacer seals an outer perimeter of the electrophoresis cassette.
The top plate assembly includes a central plate and a bottom plate, the bottom plate longer in length than the central plate. The central plate and the bottom plate are similarly rectangular in shape. The central plate has a first terminal edge or a cathode edge that is connected to a cathode buffer reservoir, and a second terminal edge or an anode edge that is connected to an debuffer reservoir. Side edges of the central plate are substantially covered by side rails that protect the side edges of the central plate during use. The side rails extend beyond the terminal ends of the central plate and connect with the side walls of the cathode and the anode buffer reservoirs to provide mechanical support to the top plate assembly.
The cathode buffer reservoir is a substantially rectangular receptacle of a sufficient depth to provide an adequate supply of buffer solution to maintain a consistent electrical current for the duration of an electrophoretic run. The cathode buffer reservoir includes a planar base connected to a body extending from the base to define the receptacle of the cathode buffer reservoir. The planar base includes a plurality of sample loading wells incorporated along a side wall of the planar base such that when the cathode buffer reservoir is connected to the cathode edge of the central plate the plurality of sample loading wells are positioned flush with the cathode edge.
The plurality of sample loading wells provides fluid communication between the cathode buffer reservoir and the molding space. The plurality of sample loading wells is defined by well walls that may be constructed of a suitable rigid, non-electrically conducting material. The well walls may include an upper terminal end that tapers to prevent sample dispensing equipment from impacting the well walls when dispensing samples into individual sample loading wells.
A mating comb is also provided in the first embodiment having a plurality of teeth or prongs. The teeth or prongs are similar in number, configuration and overall dimensions as the plurality of sample loading wells such that the mating comb is insertable into and fits flush with the plurality of sample loading wells.
The cathode buffer reservoir also includes an electrode that extends across the receptacle and provides electrical current to the cathode buffer reservoir from an external electrical source. The electrode is coupled within the cathode buffer reservoir. An electrode connector connects with the electrode and serves as a contact point for the external electrical source.
The anode buffer reservoir is a substantially rectangular receptacle of a sufficient depth to provide an adequate supply of buffer solution to maintain a consistent electrical current for the duration of an electrophoretic run. The anode buffer reservoir includes a substantially planar base. A through slot is disposed substantially centrally in the planar base. A channel is defined by the planar base and between the through slot and a side wall of the anode buffer reservoir proximate to the central plate, and is in fluid communication with the through side. The channel is also in fluid communication with an opening in the side wall. The opening is raised upward from a bottom of the side wall more than the thickness of an electrophoretic gel and less than the height of the central plate. In addition, the opening is in fluid communication with the molding space between the central plate and the bottom plate when the electrophoresis cassette is assembled.
The side wall encloses the anode buffer reservoir such that the central plate is not required to act as a fourth wall to form the receptacle of the anode buffer reservoir. The side wall includes protrusions. The protrusions increase a volume of the receptacle of the anode buffer reservoir and increase the surface area available to connect the anode buffer reservoir the central plate. The protrusions also add support for the anode buffer reservoir when the electrophoresis cassette is assembled.
The anode buffer reservoir also includes an electrode that extends across the receptacle and provides electrical current to the anode buffer reservoir from an external electrical source. The electrode is coupled within the anode buffer reservoir. An electrode connector connects with the electrode and serves as a contact point for the external electrical source.
The cathode buffer reservoir, the central plate, the anode buffer reservoir and the side rails are assembled to form the top plate assembly. The components may be adhesively connected such that the cathode buffer reservoir is adhesively connected at the cathode edge of the central plate and the anode buffer reservoir is adhesively connected to the anode edge of the central plate. Similarly, the side rails may be adhesively connected to the side edges of the central plate.
The bottom plate of the electrophoresis cassette has a length to accommodate the length of the central plate plus a width of the cathode buffer reservoir and a width of the anode buffer reservoir. The spacer is a continuous frame-type configuration seated on a top surface of the bottom plate substantially adjacent to a perimeter edge of the bottom plate. The spacer, the bottom plate and the central plate define the molding space and the thickness of the electrophoresis cassette.
The bottom surface of the central plate and the top surface of the bottom plate are substantially uniform and parallel. In addition, the spacer has a substantially uniform surface. The substantially uniform and parallel surfaces of the central plate and the bottom plate, and the substantially uniform surface of the spacer help to ensure a uniform thickness of an electrophoresis gel cast in the molding space.
To assemble electrophoresis cassette, the spacer is seated on the top surface of the bottom plate and the top plate assembly is seated on a top surface of the spacer and the top surface of the bottom plate. The assembled components are held in close proximity and alignment by clamping mechanisms, such as binder clamps, or other fasteners well known in the art.
To cast an electrophoretic gel, the electrophoresis cassette is assembled as described above. Flowable gel material suitable for molding an electrophoretic gel, such as a gel solution, is poured or injected into the anode buffer reservoir. The gel solution flows into and substantially fills the through slot, the channel and the opening of the anode buffer reservoir. The gel solution also flows beneath the anode buffer reservoir and substantially fills an area defined by a bottom surface of the anode buffer reservoir, the spacer and the top surface of the bottom plate. From the through channel, the gel solution flows into and substantially fills the molding space defined by the central plate, the spacer and the bottom plate. The gel solution flows from the molding space and into the plurality of sample loading wells and substantially fills individual sample wells. The gel solutions also flows beneath the cathode buffer reservoir and substantially fills an area defined by a bottom surface of the cathode buffer reservoir, the spacer and the top surface of the bottom plate.
The mating comb is placed in the plurality of sample loading wells to substantially displace the gel solution therein. The mating comb remains in the plurality of sample loading wells until the gel solution polymerizes. After polymerization, the mating comb is removed and individual sample loading wells are gel-free and ready for sample loading.
Thin layers of polymerized gel seal the area between the cathode buffer reservoir and the bottom plate. Thin layers of polymerized gel also seal the plurality of sample loading wells with the cathode edge of the central plate. The area beneath the anode buffer reservoir contains thin layers of polymerized gel to seal the anode buffer reservoir with the bottom plate. The through slot, the channel and the opening are substantially filled with polymerized gel to seal the anode buffer reservoir and the anode edge of the central plate. The contiguous nature of the polymerized gel forms leak-proof seals.
Upon completion of gel casting, electrophoresis of samples may proceed. The cathode buffer reservoir is filled with distilled water or a dilute salt solution to serve as a sample injection solution. The anode buffer reservoir is filled with electrolytic buffer solution of an appropriate concentration. Samples are dispensed into the plurality of sample loading wells either manually or with automated dispensing equipment. The samples are injected into the electrophoresis gel by application of a brief pulse of high voltage electrical current to the cathode buffer. The sample injection solution contained in the cathode buffer reservoir is replaced with an electrolytic buffer of an appropriate concentration. Electrical current to conduct electrophoresis is supplied by an external electrical source. Temperature control of the electrophoresis gel is supplied by a temperature control mechanism.
A second embodiment of the invention includes similar components as the electrophoresis cassette of the first embodiment except that the plurality of sample loading wells comprises a plurality of through holes machined or molded in a surface of the planar base of the cathode buffer reservoir. The plurality of through holes is positioned substantially adjacent to the side wall of the cathode buffer reservoir that is positioned flush with the cathode edge of the central plate when the electrophoretic cassette is assembled. A mating comb is provided with a plurality of teeth or prongs that are similar in number, configuration and overall dimensions as the plurality of through holes such that the mating comb is insertable into and fits flush with the plurality of through holes. Although the through holes may be of any configuration and shape, the plurality of through holes of the second embodiment are circular cylinders to facilitate ease in manufacture.
A third embodiment of the invention includes similar components as the electrophoresis cassette of the first embodiment except that the plurality of sample loading wells is arranged as staggered dual parallel linear arrays of through holes. Individual through holes of a first linear array are staggered and parallel in relation to individual through holes of a second linear array. The staggered arrangement of parallel linear arrays of through holes acts to spatially stagger sample loading to effect the technique of sample lane identification or lane tracking that is typically achieved by temporally staggering the injection of samples into the electrophoresis gel. Individual through holes may be configured as circular cylinders which permits ease in manufacturing.
A fourth embodiment of the invention provides an electrophoresis cassette similar to the electrophoresis cassette of the first embodiment except that a cathode buffer reservoir, an anode buffer reservoir and a central plate are not permanently joined into a single assembly, but rather are held together by a mechanical biasing system. The mechanical biasing system holds and maintains components of the electrophoresis cassette of the fourth embodiment in close proximity and alignment during gel casting and electrophoresis. Individual through holes of the fourth embodiment may be circular cylinders to permit manufacturing ease.
The cathode buffer reservoir includes a body and a base perimeter. The body includes a bottom planar surface with a plurality of sample loading wells incorporated with a side wall of the bottom planar surface. The body traverses the central plate and has a width equal to a length of the central plate. The body includes the mechanical biasing system that comprises spring-biased slide blocks positioned at end portions of the body for mechanically biasing the cathode edge of the central plate flush against the plurality of sample loading wells when the electrophoresis cassette is being assembled. The body also includes locating pins that downwardly protrude from the end portions to facilitate positioning of the cathode buffer reservoir relative to the bottom plate.
The base perimeter is constructed in a U-shaped configuration and is substantially flat and uniform. The base perimeter traverses the central plate and has a length substantially equal to the width of the body. The base perimeter is adhesively connected to the top surface of the central plate and positioned on the central plate such that end portions of the U-shaped configuration terminate in flush alignment with the cathode edge of the central plate. The end portions also include reference point protrusions that extend laterally at the end portions of the base perimeter to ensure the end portions are in consistent alignment with the cathode edge.
The anode buffer reservoir is substantially similar in structure and construction as the anode buffer reservoir of the first embodiment except that the anode buffer reservoir in the fourth embodiment includes locating pins that downwardly protrude from end portions of the anode buffer reservoir. The locating pins are positioned on the anode buffer reservoir in alignment with side edges of the central plate.
The anode buffer also includes a channel in a lower section of side wall of the anode buffer reservoir defined by the bottom plate and between a through slot and an opening in the side wall. The through slot is positioned substantially off-center in a planar base of the anode reservoir and substantially adjacent to the channel such that the through slot and the channel are in fluid communication. The channel is in fluid communication with the opening in the side wall. The opening is raised upward from a bottom of the first side wall more than a thickness of an electrophoretic gel contained in the electrophoresis cassette and less than the height of the central plate. The channel is positioned between the anode edge of the central plate and the through slot when the electrophoresis cassette is assembled.
The anode buffer reservoir also includes a gasket connected to are outer surface of the side wall of the anode buffer reservoir proximate to the central plate to effect a seal between the anode buffer reservoir and the anode edge of the central plate.
The spacer and the bottom plate are substantially similar in structure and construction to the spacer and the bottom plate of the first embodiment except that the spacer and the bottom plate in the fourth embodiment include features to receive the locating pins of the cathode buffer reservoir and the anode buffer reservoir. The spacer includes notches at approximately each corner to receive the locating pins of the buffer reservoirs. The bottom plate includes slots located approximately adjacent to each corner along side edges. The notches of the spacer and the slots of the bottom plate are in longitudinal alignment such that during assembly of the electrophoresis cassette the locating pins of the cathode and anode buffer reservoirs are received by the notches of the spacer and subsequently received by the slots of the bottom plate.
The electrophoresis cassette of the fourth embodiment is assembled by seating the spacer on a top surface of the bottom plate and inserting the locating pins of the cathode buffer reservoir through the notches of the spacer and into the slots of the bottom plate. The central plate with the base perimeter facing upward is seated on a top surface of the spacer. The cathode edge of the central plate is positioned flush with the plurality of sample loading wells of the body of the cathode reservoir buffer. The central plate is manually forced against the body of the cathode reservoir buffer which compresses the spring-biasing mechanisms positioned in the body of the cathode reservoir buffer, enabling the cathode edge to make firm contact with the plurality of sample loading wells. In addition, the end portions of the base perimeter are flush against and compress gaskets located at the end portions of the body of the cathode buffer reservoir to achieve leak-proof seals. The anode buffer reservoir is subsequently seated on the top surface of the spacer at the anode edge of the central plate by inserting the locating pins of the anode buffer reservoir through the notches of the spacer and into the slots of the bottom plate. When the anode buffer reservoir is connected to the bottom plate and positioned flush with the anode edge, the central plate is manually released. The spring-biasing mechanisms expand, compressing the central plate and the bottom plate between the cathode and anode buffer reservoirs, and compressing the central plate downwardly against the spacer and the bottom plate.
The electrophoretic cassette of the fourth embodiment is similarly used to cast electrophoretic gels according to the method of the first embodiment. Sample injection and electrophoresis proceed as described in the first embodiment.
A fifth embodiment of the invention includes a clamping/assembling fixture suitable for use in assembling any of the electrophoresis cassettes of the previous embodiments. The clamping/assembling fixture is a frame-type configuration with a length and a width slightly larger than the width of the electrophoresis cassettes. A plurality of fasteners is positioned on at least three sides of the clamping/assembling fixture to hold the central plate, the spacer and the bottom plate, and optionally at least one buffer reservoir, in close proximity and alignment.
A sixth embodiment of the invention includes an electrophoresic cassette similar to the electrophoresis cassette of the first embodiment except that a modified spacer with a U-shaped frame, a cathode blank and a mechanical biasing system are employed. The modified U-shaped spacer is seated on the top surface of the bottom plate such that a continuous portion of the modified spacer is under the anode buffer reservoir and end portions of the spacer terminate on the top surface of the bottom plate at a position flush with the cathode buffer reservoir.
The cathode blank replaces the cathode buffer reservoir during gel casting and substantially fills a space on the top surface of the bottom plate occupied by the cathode buffer reservoir. The cathode blank acts as a template to restrict flow of the gel solution to the cathode edge of the central plate. The central plate, the modified U-shaped spacer, the bottom plate and the cathode blank define a molding space. The cathode blank is removed and replaced with the cathode buffer reservoir after completion of gel casting. The cathode buffer reservoir is positioned flush with the cathode edge of the central plate.
The mechanical biasing system includes systems such as those disclosed in U.S. Pat. Nos. 5,242,568, 5,228,971 and 5,137,613, incorporated herein by reference. The mechanical biasing system holds and maintains assembly components in close proximity and alignment during gel casting and electrophoresis. The mechanical biasing system biases the cathode buffer reservoir against the cathode edge of the central plate to position the plurality of sample loading wells flush with the cathode edge. The mechanical biasing system similarly biases the anode buffer reservoir against the central plate to position the anode buffer reservoir flush with the anode edge of the central plate. In addition, the mechanical biasing system ensures downward biasing of the central plate against the spacer and the bottom plate.
The electrophoresis cassette of the sixth embodiment is used to cast an electrophoretic gel according to the method of the first embodiment except that the modified U-shaped spacer and the cathode blank are employed during gel casting. The molding space defined by the bottom plate, the modified U-shaped spacer, the central plate and the cathode blank substantially fills with the gel solution. After polymerization, the cathode blank is removed. The electrophoretic gel polymerized in the molding space terminates in flush alignment with the cathode edge of the central plate. The cathode buffer reservoir is seated on the top surface of the bottom plate with the plurality of sample loading wells positioned flush with the cathode edge and a terminal edge of the electrophoretic gel. Sample injection and electrophoresis then proceed as described in the first embodiment.
A seventh embodiment of the invention includes an electrophoresis cassette similar to the electrophoresis cassette of the sixth embodiment except that the cathode blank and the modified U-shaped spacer are not used. A spacer with a continuous frame-type configuration is employed and seated on the top surface of the bottom plate. The cathode buffer reservoir is seated on a top surface of the spacer and includes recesses on a bottom surface. The recesses have approximately the same width and depth as the spacer and are positioned on the bottom surface of the cathode buffer reservoir in direct alignment with the spacer. When the cathode buffer reservoir is seated on the top surface of the spacer, the recesses receive portions of the spacer on which the cathode buffer reservoir is seated. This arrangement permits the cathode buffer reservoir to be seated substantially flush with the top surface of the bottom plate.
The electrophoresis cassette of the seventh embodiment is used to cast an electrophoretic gel according to the method of the first embodiment except that the cathode buffer reservoir with the recesses as described above is employed. The gel solution is substantially prevented from flowing into an area under the cathode buffer reservoir, although extremely small amounts of the gel solution permeate under and into the area under the cathode buffer reservoir. Polymerization of small amounts of gel solution seal the bottom of the cathode buffer reservoir to the top of the bottom plate. Sample injection and electrophoresis then proceed as described in the first embodiment.